Glioblastoma multiforme (GBM) is a highly lethal brain cancer that is refractory to virtually all treatment modalities. The targeting of the receptor tyrosine kinase (RTK) EGFR, an important pathogenetic lesion in GBM, has elicited only modest and transient responses in a subset of patients with intact PTEN. We recently discovered that multiple RTKs are co-activated in GBM and other tumors, such that redundant nputs drive and maintain downstream signaling to limit the efficacy of therapies targeting single RTKs. Given the capacity to profile RTK activation patterns in primary cancers and the availability of numerous RTK inhibitors, these observations hold significant clinical potential. This proposal will further characterize the requirements for RTK cooperation in gliomagenesis through the following specific aims: Aim 1: In vitro RTK cooperativitv analyses in normal human astrocvtes. This aim seeks to examine the biological and biochemical impact of various GBM-relevant RTKs in a well-defined primary human cell culture based model. Specifically, immortalized normal human astrocyte cells will be engineered with one or more RTKs observed to be activated in primary human patient samples. These RTKs will be expressed either inducibly or constitutively and their specific and cooperative effects on cell viability, migration, anchorage-independent growth, and downstream biochemical signaling will be monitored in vitro. Aim 2: In vivo RTK cooperativitv analyses in orthotopic xenografts. Because tumor microenvironment can influence RTK signaling, this aim seeks to build on the identification of key RTKs in Aim 1 through an in depth in vivo analysis of the tumorigenic impact of RTKs, singly or in combination, on the malignant transformation of human astrocytes or murine neural stem cells in an orthotopic setting. By engineering these cells with inducible RTK alleles, it will also be possible to assess the biological and biochemical impact of extinguishing expression of one or more RTKs once tumors have been established. Tumor phenotype will be carefully monitored for angiogenesis, invasion, and necrosis in an effort to establish genotype-phenotype relationships for each RTK alone or in combination. Aim 3: RTK function in tumor stem cells. It has been proposed that durable cures for cancer may require targeting tumor stem cells. On the basis of this hypothesis, we will profile RTK co-activation patterns in tumor initiating cells vs. bulk tumor cells from primary human GBM samples. To assign biological relevance to these patterns, pharmacological or lentiviral shRNA approaches will be used to assess the anti-tumor impact of extinguishing one or more activated RTKs in the tumor initiating cells. Public Health Relevance: These studies will add to our knowledge of the molecular lesions driving GBM and enable a more accurate pre-clinical analysis of drugs targeting RTKs active in this lethal disease.